Fig. 3
Activation of p21 in umbilical cord derived mesenchymal stem cells (UC-MSC) responsible for augmented apoptosis induction by cyclic mechanical stretch (CMS) and hyperoxia (HOX). (A) Representative western blot analysis of n=7 independent UC-MSC cultures experiments for p21 activation after 24h exposure to CMS and HOX 80%. CMS and HOX induced the accumulation of p21 whereas more pronounced for HOX and CMS+HOX, n = 7 MSC cultures. The order of samples was rearranged for the clearness of presentation without any further manipulation (indicated by separated boxes). The bar graphs show the densitometry results expressed as the ratio of adjusted total band volume normalized to ßActin and control. (B) Representative western blot analysis of n = 7 independent UC-MSC cultures of sip21 transfected cells show inhibited accumulation. The order of samples was rearranged for the clearness of presentation without any further manipulation (indicated by separated boxes). The bar graphs show the densitometry results expressed as the ratio of adjusted total band volume normalized to ßActin and unexposed UC-MSC for the n = 7 independent UC-MSC cultures. (C) Representative flow cytometry analysis of n = 7 independent UC-MSC cultures of each intervention group shows increase of apoptotic cell fraction in sip21 transfected UCMSC for each treatment with CMS and/or HOX 80%. Live/dead staining via Annexin V/SYTOX Blue flow cytometry analysis, absolute cell death (%) = dead cells / frequency of single cells. (D) Percentage of absolute apoptosis induction following selective and combined treatment with CMS and/or HOX (HOX 80%) of mock or p21 siRNA transfected UC-MSC by for the n = 7 independent UC-MSC cultures. Data are expressed as box plot with median and interquartile range, *p < 0.05, **p < 0.01, by paired t-test, comparing exposures to the control situation with specific siRNA against p21 to mock transfection respectively. All full-length blots/gels from this figure are presented in Supplementary Fig. S1.
